Suboptimal sperm motility accounts for nearly 30% of all human infertility. As such, a better understanding of the biochemical mechanisms regulating sperm motility may provide therapeutic options for infertile couples. We have recently identified a gene product (ROPN1) that appears to be critical for both sperm motility and fertility. ROPN1 is highly expressed in testis and sperm, and mice lacking ROPN1 have impaired sperm motility and are subfertile or infertile. The expression level of ROPN1 is significantly lower in asthenozoospermic men than in normozoospermic controls. ROPN1 was originally identified as a protein that binds to rhophilin, a binding partner of Rho. Using a yeast two-hybrid analysis, we discovered that ROPN1 also binds to sperm A-kinase anchoring proteins (AKAPs). ROPN1 contains a dimerization/docking domain similar to the regulatory subunit of protein kinase A (PKA). However, outside the docking domain, ROPN1 bears little or no similarity to PKA suggesting they have distinct functions. Two observations suggest that ROPN1 is required for flagellar motility: 1) adding peptides (Ht31) that disrupt the interaction between AKAPs and ROPN1 results in altered protein phosphatase 1 (PP1) activity, GSK3 phosphorylation and arrested sperm motility, and 2) sperm from mutant mice lacking ROPN1 (Ropn1-/-) have reduced motility. Thus, ROPN1 appears to be a key molecule located at the intersection between the cAMP and Rho pathways. Although many articles have been published on the role of cAMP/PKA in sperm, the literature on Rho in sperm is relatively sparse. Inactivation of Rho-GTPase has been shown to reduce mammalian sperm motility and we have recently shown that sperm contain all the protein components of the Rho signaling pathway. Inhibition of PP1, a downstream effector in the Rho pathway, dramatically alters sperm motility. However, to our knowledge, no one has measured the activity of Rho-GTPase from sperm. The goal of this project is to determine if Rho is a critical component of the biochemical machinery regulating sperm motility and if ROPN1 functions as part of this pathway. Successful completion of this proposal will enhance our knowledge of this important regulatory mechanism and thus may facilitate development of treatments for male infertility. PUBLIC HEALTH RELEVANCE: The goal of this proposal is to enhance knowledge of signaling pathways that regulate flagellar function, which is essential to defining normal male fertility. This increased understanding of normal processes should allow for more precise diagnoses of the root cause(s) of infertility, and will also provide more targets for treatment and rational drug design. Commonalities between flagella and cilia in function, protein expression and signaling pathways suggest that these findings may also have wider implications in diagnosis and treatment of ciliary diseases.